A major mechanism for regulating the activity of biogenic amine neurotransmitter transporters, the important targets for therapeutic antidepressants and psychostimulant drugs of abuse, is through modulation of the abundance of functional transporters at the plasma membrane. For the dopamine transporter (DAT) we have found that regulated internalization proceeds through one of two apparently distinct mechanisms. The first pathway is stimulated by activation of protein kinase C (PKC) and appears to be mediated by a clathrin-coated pit mechanism. The second pathway is activated by exposure to amphetamine-like drugs, is not affected by PKC antagonists and does not appear to proceed through a clathrin-dependent pathway. We hypothesize that this distinct action of amphetamine and other amphetamine-like drugs may contribute to the unique features of their addictive properties. Internalization of the DAT by the two pathways can be observed to different extents in various systems, including neuronal cultures, catecholaminergic cell lines and other in vitro models. The aims of this research proposal are to define components of the internalization machinery which are selectively mobilized by the PKC- and amphetamine-induced pathways and to elucidate the cellular mechanisms that regulate DAT internalization in dopamine neurons. High-resolution confocal imaging will be employed to identify the primary endocytic pathways for both PKC- and amphetamine-stimulated internalization mechanisms in model cell lines and in primary cultures of mesencephalic dopamine neurons. siRNAi techniques will be employed to establish the two distinct mechanisms of transporter internalization. Two neuropeptide systems, the orexins and CRF, modulate dopaminergic neurotransmission in vivo by coupling to PKC signaling in dopamine neurons, and appear to play a critical role in cocaine-induced psychomotor sensitization. We will also determine to what extent these endogenous neuropeptides and other regulators activate or modulate PKC- and amphetamine-mediated DAT trafficking in dopamine neuron cultures. The goal of these studies is to more precisely define the major machinery regulating DAT activity and to understand how these pathways modulate the actions of therapeutic and abused drugs that target biogenic amine transporters.